Study of Some Antioxidant Parameters in Mice Livers Affected with Urtica pilulifera Extracts

MATERIALS AND METHODS

Preparation of Plants
Three plant parts (herbs, roots and seeds) were exhaustively extracted using two solvents; petroleum ether (80-100%) (PE) and 20% aqueous methanolic (AM) extracts; P.E. crude extracts prepared by continuous extraction using soxhlet apparatus while methanolic extracts were prepared by soaking with shaking for plant powders of each organ separately, then each extract was filtered to remove cellular residues. The extracts were concentrated under reduced pressure till free solvents. The different residues were used for experiment.

Animals
Male swiss albino mice (8-9 weeks old), bred in the animal house of National Research Center, were used. The animals were maintained under normal conditions (12 h light/dark cycle) and were fed with pelleted standard laboratory mice feed and tap water.

Experimental Procedure
A total of 49 mice were used, divided into the following groups: Group I (n = 7) was injected interpretoneal with saline, daily for 10 days. This group was designated as a control group. Group II, III and IV (each of 7 animals) were treated with 50 ppm (0.25 mL¹ mouse) of petroleum ether extract of U. pilulifera (herbs, roots and seeds) through interpretoneal injection daily for 10 days. Group V, VI and VII (each of 7 animals) were treated with 50 ppm (0.25 mL¹ mouse) of methanol extract of herb, root and seed through interpretoneal injection daily for 10 days. This treatment is repeated with high concentration (200 ppm).

Preparation of Subcellular Fractions from the Liver
Mice were killed by cervical dislocation. Liver homogenates were prepared in ice-cold bidistilled water. The homogenate was centrifuged at 3000 rpm in cooling centrifuge (-4.C) for 10 min. The supernatant was used for determination of NP-SH and different enzymatic activities.

Determination of NP-SH Group
The level of sulfhydryl groups was determined using 5,2-dithiobis-2-nitrobenzoic acid (DTNB) according to Neugebauer et al. (1995).

Determination of Lipid Peroxidation (LPO)
LPO of liver microsomes was determined spectrophotometrically as the number of nmoles of malondialdehyde (MDA) formed per mg protein at 532 nm (Oktay et al., 2003).

Assay of Glutathione S-transferase (GST)
It was assayed using 1-chloro-2, 4- dinitrobenzene (CDNB) as substrate (Habig et al., 1974). The substrate was previously dissolved in the least amount of absolute ethyl alcohol. The formation of conjugated derivatives was followed spectrophotometrically at 340 nm as a function of time.

Determination of Protein
Microsomal and cytosolic proteins were determined using the method of Bradford (1976).

Assay of Glutathione Reductase (GR)
GR was assayed spectrophotometrically with slight modification according to Calberg and Mannervik (1974).

Assay of Glutathione Peroxidase (GSHPx)
GSH peroxidase activity was determined as described by Lebedev et al. (2001).

Assay of Superoxide Dismutase (SOD)
Measurement of SOD activity was carried out spectrophotometrically (Folhé and Otting, 1984).

Statistical Analysis
Results are expressed as mean ± SD. Statistical analysis was determined using ANOVA following Dunnet’s t-test. Values showed a significant difference from those of control groups. The SPSS 10.0 (Statistical Program for Social Sciences) was used for statistical analysis (Campbell, 1989).

Results

Antioxidant Activity of Aqueous Methanolic and Peterolum Ether Extracts at 50 ppm
Table 1 and 2 indicate that all six U. pilulifera extracts significantly enhanced the protein synthesis in mice liver and enhanced glutathione reductase activity but the glutathione transferase activity reduced than control group using aqueous methanolic extracts of herbs and roots. Superoxide dismutase activity was significantly enhanced by using all extracts.

Table 1:	Effect of Urtica pilulifera methanolic extracts at 50 ppm on antioxidant parameters in mice liver homogenates

Table 2:	Effect of Urtica pilulifera petroleum ether extracts at 50 ppm on antioxidant parameters in mice liver homogenates

Table 3:	Effect of Urtica pilulifera methanolic extracts at 200 ppm on antioxidant parameters in mice liver homogenates
Conc: Concentration, G: Glutathione, P: Protein, SOD: Superoxide dismutase, F: ratio of one way ANOVA, P: value of ANOVA significant <0.05

Antioxidant Activity of Aqueous Methanolic Extracts at 200 ppm
All estimated protein concentration and antioxidant parameters were enhanced specially with herbal extract, the protein concentrations measured in liver homogenate of mice treated with 200 ppm aqueous methanolic extracts were enhanced by 25.97, 75.86 and 78.7% for herbs, roots and seeds, this increment of protein synthesis was in parallel with induction of enzyme levels.

Table 4:	Effect of Urtica pilulifera petroleum ether extracts at 200 ppm on antioxidant parameters in mice liver homogenates
Conc: Concentration, G: Glutathione, P: Protein, SOD: Superoxide dismutase, F: ratio of one way ANOVA, P: value of ANOVA significant <0.05

Table 3 shows that, glutathione concentrations determined in mice liver homogenate were significantly increased specially in case of mice treated by herbal extracts (299.2, 84.2 and 22.5% for herb, root and seeds, respectively).Glutathione transferase activity was elevated by 285.76, 118.69 and 181% using herbs, roots and seeds extracts, respectively. The glutathione reductase activity was enhanced with animal treated by herbs extract only (36.69%) whereas roots and seeds extracts were reduced by 24.88 and 20.69%, respectively. Glutathione peroxidase has the same manner which induced by herbal extract (68.3%) and reduced by the other extracts; roots and seeds (-40.14 and -43.66%, respectively). The superoxide dismutase (SOD) levels were increased by (232.8%) using extract of herb while the other extracts (roots and seeds extracts) caused lowered increments by 31.25 and 64.84%, respectively. The enhancement of antioxidant parameters caused high reduction in lipid peroxidation and the highest reduction was caused by aqueous methanolic herb extract which represented 81.8% and followed by roots and seeds extract (69.86 and 58.39%, respectively).

Antioxidant Activity of Petroleum Ether Extracts at 200 ppm
Table 4 shows that liver protein concentrations in mice treated by petroleum ether extract were significantly increased by 132.76, 120.97 and 64.7% for herb, roots and seeds extract, respectively. The glutathione concentrations were enhanced by 66.8, 80 and 126.8% for herb, roots and seeds extract treatments, at the same time the glutathione transferase activities were induced by 169.57 , 104.35% for herb and roots extracts treatments while it decreased by seeds extract treatment (-10.14%). The glutathione reductase activities were enhanced by different levels; 59.92% for herbs extract treatment, 25.15% for roots extract treatment and 10.22% with seeds extract treatment. On the other hand, glutathione peroxidase has high significant increase by 187.72% when animals treated by petroleum ether extract of herb, but roots and seeds petroleum ether extract did not have the same effect which caused enhancement by 34.04 and 15.09%, respectively.

Data of superoxide dismutase activity in liver tissue of treated animals showed an increment using herb petroleum ether extract (26.32%) whereas they decreased by 78.95 and 52.63% in animal treated by roots and seeds pet. ether extracts. Herb petroleum ether extract has a strongest effect on antioxidant parameters, it inhibited lipid peroxides production (-31.2%) but the other two extracts did not have the same activity, they enhanced lipid peroxides by 12.0 and 52.8% for roots and seeds extracts, respectively.

DISCUSSION

In this study, application of the petroleum ether and hydro alcoholic extracts of Urtica pilulifera (U.P) to mice showed a marked effect on protein synthesis, some hepatic biotransformation enzyme systems and antioxidant enzymes. The intraperitoneal injection of U. pilulifera extracts (0.735 and 2.94 mg kg¹day for 10 days) had discernible effects on the tested parameters.

Protein concentration was increased by different extracts in the order of herb>root >seed by using PE extracts and take the opposite direction by using methanol (Table 1 and 2). This may be due to presence of a procial amount of active compounds that enhance protein synthesis. This process also differs according to content of active compounds in different part of plants which affects some hepatic biotransformation and antioxidant enzymes. The enhancement of protein synthesis is also dose dependent (Table 1 and 3). The decrease of lipid peroxidation by U. pilulifera suggests a role of cytoprotection as well as prooxidant and peroxisomes induced membrane dangerous.

The findings are coincided with Ozen and Korkmaz, 2003 who used hydroalcoholic herb extract of U. dioica by 50 mg and 100 mg kg¹day. Table 1 and 2 showed that U. P. has a high antioxidant activity although it was used in low concentration, this indicates high reducing power activities of the plant. The antioxidant activity of putative antioxidants have been attributed to various mechanisms, among which are prevention of chain initiation, binding of transition metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and radical scavenging (Samur et al., 2001).

GST, which plays an important role in conjugating the metabolites resulting from the action of cytochrome P450, with endogenous ligands (reduced glutathione) favoring their elimination from the body of organisms, is a critical detoxification enzyme (Hartman and Shankel, 1990). It has already been reported that glutathione S-transferase induces protection against various cytotoxic, mutagenic and carcinogenic chemicals (Ketterer, 1988). The study showed that GST activity increases (in common) by using different extracts and this increase differs in its magnitude according to dose and parts of plants. The antioxidant enzymes in mice liver is enhanced with using leaf extracts (PE and AM) but decrease in roots and seeds. This may be due to quinching activity of different parts of plants and hydrogen donating ability of separate compounds (Gülçin et al., 2004). The phytochemicl investigation of the plants showed the presence of flavonoids, coumarins, some sterols, alkaloids and hydrocarbons by aqueous methanol extract meanwhile sterols, fatty acids and terpenes were extracted by petroleum ether. Flavonoids, phenolic acids and some terpenoids possess antioxidant activities in different mechanisms which gave the induction of different antioxidant estimated parameters.

On the basis of the results of this study, it is clearly indicated that U. pilulifera has a powerful antioxidant activity against various oxidative systems in vivo moreover; it can be used as accessible source of natural antioxidants and as a possible food supplement or in pharmaceutical industry. In addition, free radicals have been demonstrated to be a contributing factor in the tissue injury and modulation of the pain, phenolic compounds which present in plants appear to be responsible for the antioxidant activity of extracts. Some studies have revealed that the antioxidants melatonin and β-carotene potentiate the antinociceptive responses (Ruiz-Larrca, 1997).